The strategies under Goals above involve several steps that together comprise a push-pull approach. First, we optimize the antigen to improve immunogenicity by epitope enhancement, increasing affinity for MHC. We have done this for 2 new prostate cancer antigens, TARP and POTE. We fully accrued a phase I/II TARP clinical trial in D0 prostate cancer patients with rising PSA levels using a TARP peptide that we epitope-enhanced to improve HLA-A2 binding and a second high affinity one we mapped. The slope of PSA rise significantly decreased among &gt; 70% of the first 39 patients enrolled (p = 0.015 at 24 wks; p = 0.0076 at 48 wks), suggesting slowing of cancer growth. The second step is to push the response with molecular adjuvants, such as cytokines, Toll-like receptor (TLR) ligands and NKT agonists, to improve not only the quantity but also the quality of the response. We published that IL-15 is an important mediator of CD4 T cell help for CD8 T cells and also that IL-15 increased the avidity of the CD8 T cells, needed for effective clearance of virus or tumor cells. We translated this to humans showing that IL-15 could substitute for CD4 help to induce a primary in vitro CD8 T cell response of na?ve T cells whereas IL-2 could not, and restored alloresponsiveness of CD8 T cells from HIV-infected patients to normal levels. We also found that IL-1beta as adjuvant could enhance CD8 T cell responses and skew CD4 help to Th17. We found surprisingly that the Th17 CD4 cells were not good helpers for a CD8 T cell response as measured by IFNgamma production, but rather they skewed the CD8 response to IL-17 production through an effect on DCs.We also investigated TLR ligands as adjuvants, as these can mature DCs and induce production of cytokines like IL-12 and IL-15. We published that a synergistic triple TLR ligand combination induces more effective protection against virus infection not by increasing T cell quantity, but by improving quality by inducing higher avidity T cells, and more IL-15 production. We tested the combination of triple TLR ligands, IL-15, both or neither as vaccine adjuvants in a peptide-prime, MVA-boost mucosal vaccine for SIV in macaques, challenging intrarectally with SIVmac251. Only macaques receiving both showed partial protection. In the adaptive immune arm, only polyfunctional CD8 T cells specific for SIV antigens, but not total specific T cells, correlated with protection. In the innate immune arm, the adjuvants induced long-lived innate protection by APOBEC3G. These adjuvants also increased CD4 cell preservation in the gut, independent of viral load. Thus, molecular adjuvant vaccine strategies inducing both innate and adaptive immunity may be the most efficacious. The third step is to target the immune response to the relevant tissue, the mucosa in the case of HIV. We found that homing to the large intestine is governed in part by DCs from colon patches, using a mechanism involving alpha4beta7 but not CCR9, distinct from that in the small intestine. We are identifying chemokines to selectively target T cells to the colon. We also just published a novel nanoparticle approach to vaccine delivery to the large intestine, using vaccine nanoparticles coated with Eudragit FS30D to allow oral delivery and release of the particles primarily in the large intestine, bypassing the stomach and small intestine. This effectively substituted for intrarectal delivery to protect against rectal or vaginal viral challenge. Moreover, the novel approach allows selective oral delivery to the small or large intestine depending on the Eudragit formulation, making it possible to distinguish the effect of antigen delivery to these compartments for the first time. We found that delivery to the small intestine, in contrast to delivery to the large intestine, does not induce colorectal or vaginal immunity, but does induce immunity in the small intestine. We have recently adapted this approach to non-human primates in an AIDS vaccine. 2/7 animals so immunized were protected from acquisition of SHIVsf162P4 moderate dose rectal challenge, although immune correlates of protection are not yet clear. We have also been studying the induction of CD8 T cell responses in the vaginal mucosa and have shown that local mucosal CD8 T cells can be induced to respond and proliferate directly in the vaginal mucosa, apparently independently of the draining lymph nodes, contrary to common belief.The fourth step is to remove the brakes, i.e., block negative regulatory mechanisms that inhibit immunity. We previously discovered a new immunoregulatory pathway involving NKT cell suppression of tumor immunity, dependent on IL-13 and TGF-beta. We found that type I NKT cells (using an invariant TCRalpha chain) protected, whereas type II NKT cells (using diverse TCRs) suppressed immunity. Moreover, type I & type II NKT cells cross-regulated each other, defining a new immunoregulatory axis. The balance along the NKT axis could influence subsequent adaptive immune responses. We are researching tumor lipids that stimulate NKT cells, markers to identify type II NKT cells, the mechanisms of suppression and also the relationship between suppressive NKT cells and CD25+ Foxp3+ T regulatory cells. We observed two different regulatory cells (Tregs & type II NKT) suppressing immunity to the same tumor simultaneously but independently for the first time, and found that a third T cell, the type I NKT cell, can determine the balance between these two regulatory cells, regulating the regulators. As humans with cancer often have a deficiency of type I NKT cell function, they may require blockade of both T regs and type II NKT cells to reveal tumor immunity. We also recently developed a way to make sulfatide-loaded CD1d dimers that can stain type II NKT cells, allowing detection of these otherwise elusive cells that have been hard to study. Conversely, stimulating with a type I NKT cell agonist can protect against tumors. We discovered that a b-mannosyl analog of a-galactosylceramide, unlike other beta-linked sugar ceramides, is also protective. However, its mechanism of protection against cancer is different from that of the classic a-GalCer, being dependent on TNF-alpha and nitric oxide synthase rather than on interferon-gamma, and it synergizes with a-GalCer. We have now also found that it does not induce the degree of anergy found after a-GalCer injection, so 2 months after b-ManCer treatment, in contrast to a-GalCer, b-ManCer and a-GalCer both protect. B-ManCer also stimulates human NKT cells. This first representative of a new class of NKT cell agonists should be translated to human cancer therapy. A key mediator of the NKT regulatory pathway and an important regulator of T regulatory cells is TGFbeta. We found that blockade of TGFbeta can protect against certain tumors in mice, and can synergize with anti-cancer vaccines in 2 mouse models. The protection is dependent on CD8 T cells. We have translated this into a clinical trial of a human anti-TGFbeta monoclonal antibody in a CRADA with Genzyme, in melanoma. The phase I study showed some activity (one long partial remission, 3 mixed responses and 2 cases of stable disease among 22 patients). Finally, we found that an adenovirus vaccine expressing the extracellular and transmembrane (ECTM) domains of HER-2 can cure large established mammary cancers and lung metastases in mice. The mechanism involves antibodies that inhibit HER-2 function, rather than T cells, and is FcR independent, unlike Herceptin, and may work in Herceptin failure because the mechanism is different. We have now made a similar cGMP recombinant adenovirus expressing the human HER-2 ECTM domains to carry out a clinical trial in cancer patients, have just received approval from the SRC and IRB, and are now submitting an IND.